it is essential that the modified protein is fully characterized and
that the ratio of probe to protein should be determined. It
should also be demonstrated that the modification does not
affect any biological activity of the protein. Finally, equilibrium
binding measurements should be performed to determine the
affinity of the modified protein for the ligand and this should
be compared with that of the native protein. This can be usually
done using suitable fluorescence-based competition or displacement experiments or by using some of the other biophysical techniques described in this book.
2. If the fluorescence of the labeled protein does not change upon
binding, it may be possible to study the interaction using
anisotropy measurements. Such measurements require an
instrument equipped with a polarizer filter in the excitation
path which can be rotated to give either vertically or horizontally polarized light. Measurements are best made in what is
known as the “T” format, with two detection photomultipliers
equipped with polarizers positioned at right angles to the
incident light direction for measurement of the intensity of
the emitted light polarized parallel (I k ) and perpendicular
(I ⊥ ) to the plane of polarization of the exciting light. The
two photomultipliers will respond differently to the parallel
and perpendicular light and must first be normalized. This is
done by exciting the fluorophore with horizontally polarized
light and adjusting the high voltage on each photomultiplier so
that they give the same output signal.
The fluorophore is excited with vertically polarized light,
and the intensity of the emitted light polarized parallel (I k ) and
perpendicular (I ⊥ ) to the plane of polarization of the exciting
light is recorded. The total fluorescence intensity is given by
(I k + 2I ⊥ ), and the anisotropy is calculated as r ¼ (I k À I ⊥ )/
(I k + 2I ⊥ ). The anisotropy is related to the fluorophore’s
rotational correlation time (τ c ) by the equation r ¼ r o /
(1 + τ/τ c ), where r o is the limiting anisotropy of the fluorophore and τ is its excited state lifetime. Anisotropy measurements are particularly appropriate in the study of the binding of
small fluorescent ligands to large macromolecules because τ c is
related to size and such reactions will therefore generally be
accompanied by large increases in anisotropy. However,
because anisotropy can be measured with high precision, it is
also possible to use this approach using proteins labeled with a
fluorophore.
3. The reciprocal of k obs is called the relaxation time, or time
constant, τ, of the system and is the time taken for the signal
to change from S 0 to (S eq À (S eq À S 0 )/e). Although k obs and
τ
À1 are identical, the former is generally used to describe
100
Stephen R. Martin and Maria J. Schilstra
that the ratio of probe to protein should be determined. It
should also be demonstrated that the modification does not
affect any biological activity of the protein. Finally, equilibrium
binding measurements should be performed to determine the
affinity of the modified protein for the ligand and this should
be compared with that of the native protein. This can be usually
done using suitable fluorescence-based competition or displacement experiments or by using some of the other biophysical techniques described in this book.
2. If the fluorescence of the labeled protein does not change upon
binding, it may be possible to study the interaction using
anisotropy measurements. Such measurements require an
instrument equipped with a polarizer filter in the excitation
path which can be rotated to give either vertically or horizontally polarized light. Measurements are best made in what is
known as the “T” format, with two detection photomultipliers
equipped with polarizers positioned at right angles to the
incident light direction for measurement of the intensity of
the emitted light polarized parallel (I k ) and perpendicular
(I ⊥ ) to the plane of polarization of the exciting light. The
two photomultipliers will respond differently to the parallel
and perpendicular light and must first be normalized. This is
done by exciting the fluorophore with horizontally polarized
light and adjusting the high voltage on each photomultiplier so
that they give the same output signal.
The fluorophore is excited with vertically polarized light,
and the intensity of the emitted light polarized parallel (I k ) and
perpendicular (I ⊥ ) to the plane of polarization of the exciting
light is recorded. The total fluorescence intensity is given by
(I k + 2I ⊥ ), and the anisotropy is calculated as r ¼ (I k À I ⊥ )/
(I k + 2I ⊥ ). The anisotropy is related to the fluorophore’s
rotational correlation time (τ c ) by the equation r ¼ r o /
(1 + τ/τ c ), where r o is the limiting anisotropy of the fluorophore and τ is its excited state lifetime. Anisotropy measurements are particularly appropriate in the study of the binding of
small fluorescent ligands to large macromolecules because τ c is
related to size and such reactions will therefore generally be
accompanied by large increases in anisotropy. However,
because anisotropy can be measured with high precision, it is
also possible to use this approach using proteins labeled with a
fluorophore.
3. The reciprocal of k obs is called the relaxation time, or time
constant, τ, of the system and is the time taken for the signal
to change from S 0 to (S eq À (S eq À S 0 )/e). Although k obs and
τ
À1 are identical, the former is generally used to describe
100
Stephen R. Martin and Maria J. Schilstra